There is growing concern worldwide that extensive use of antibiotics is resulting in the development of antibiotic resistance among pathogenic bacteria. In particular, antibiotic overuse in livestock feeds compromises the effectiveness of current therapies via dissemination of antibiotic resistance genes to both disease-causing and commensal microorganisms (Yan and Gilbert, 2004, Adv. Drug Deliv. Rev. 56, 1497-1521; DuPont, 2007, Clin. Infect. Dis. 45, 1353-1361).
Over 80% of the antibiotics produced in the United States are administered in swine, poultry and cattle farming. In addition to their intended use as therapeutics, antibiotics are administered throughout the life of food producing animals, even in the absence of infection to promote animal growth and improve feed efficiency (Witte, 1998, Science 279, 996-997; Mellon et al., 2001, Hogging it: estimates of antimicrobial abuse in livestock. Union of Concerned Scientists: Cambridge, Mass.). These growth-promoting antibiotics are applied at sub-therapeutic concentrations, establishing the conditions for development of resistance to antibiotics. Alarmingly, many of the antibiotics used in agriculture have also been listed as critically important for human health by the World Health Organization. Humans depend on many of these same antibiotics as a first line of defense against pathogens like Escherichia coli O157:H7, Salmonella typhimurium, Staphylococcus aureus, Streptococcus, and Pseudomonas aeruginosa (FAO, 2007, OIE, 2008: Joint FAO/WHO/OIE Expert meeting on critically important antimicrobials, in Report of a meeting held in FAO, Rome, Italy, pp 26-30).
Enterococci are commensal organisms that form part of the normal intestinal flora of humans and animals. However they are fast emerging as pathogens causing serious and life threatening hospital borne infections. Over the last three decades enterococcal strains have evolved to resist in effect all antibiotics, including vancomycin, long considered an antibiotic of last resort for many infections (Cattoir and Leclercq et al., 2013, J Antimicrob Chemother. 68:731-42). Antibiotic-resistant enterococci are hard to treat and account for approximately 110,000 urinary tract infections, 25,000 cases of bacteremia, 40,000 wound infections, and 1,100 cases of endocarditis annually in the USA (Huycke et al., 1998, Emerg Infect Dis. 4:239-49). Enterococci are the leading cause of surgical site infection, the second leading cause of nosocomial infection, the second most common pathogen for nosocomial bacteremia and bloodstream infections, and the third leading cause of urinary tract infections (Deshpande et al., 2007, Diagn Microbiol Infect Dis. 58:163-170; Chou et al., 2008, J Microbiol Immunol Infect. 41: 124-129). The potential association between the use of broad-spectrum antibiotics and the increasing incidence of atopic and autoimmune diseases is a particular cause for concern.
One promising alternative to traditional antibiotic molecules are antimicrobial peptides (AMPs). AMPs are small, often positively-charged, peptides with high antimicrobial activity. The activity of AMPs can be broad, efficiently acting on many Gram-positive and Gram-negative bacteria species. There are however AMPs with very specific activity, targeting one particular bacteria species or even a specific subspecies of a given genus (Hancock and Lehrer, 1998, Trends Biotechnol. 16, 82-88; Ganz and Lehrer, 1999, Mol. Med. Today 5, 292-297; Kokryakov et al., 1993, FEBS letters 327, 231-236; Cotter et al., 2013, Nat. Rev. Microbiol. 11, 95-105; Brogden et al., 2005, Nat. Rev. Microb. 3, 238-250; Man et al., 2006, Curr. Opin. Pharmacol. 6, 468-472).
The current production, purification and delivery methods available for these peptides have numerous limitations. For example, solid state peptide synthesis and peptide production and purification from cell culture are both costly and time consuming (Marr et al., 2006, Curr. Opin. Pharmacol. 6, 468-472; Nilsson et al., 2005, Annu. Rev. Biophys. Biomol. Struct. 34, 91-118; Gräslund et al., 2008, Nat. Methods 5, 135-146). Additionally, the subsequent targeted delivery of active amounts of these compounds can be challenging. Generally, AMPs cannot be administered orally as they are quickly degraded before they are able to reach their target. AMPs cannot be administered systemically either, as they are rapidly identified and targeted for clearance by the immune system before they can reach the site of infection (Man et al., 2006, Curr. Opin. Pharmacol. 6, 468-472). Moreover, high peptide concentrations are required to achieve a therapeutic effect which would be cost-prohibitive and would, more importantly, cause severe toxic side-effects. Taken together, these limitations have thus far stifled the development of AMP-based therapeutics (Marr et al., 2006, Curr. Opin. Pharmacol. 6, 468-472).
In recent years probiotic bacteria have emerged as useful tools for effectively boosting overall human and animal health (Oelschlaeger et al., 2010, Int. J. Med. Microbiol. 300, 57-62). Probiotics are typically Gram-positive, bile-resistant, bacteria that either colonize or transiently inhabit the gastrointestinal (GI) tract of a host. When administered in adequate amounts they confer health benefits by improving nutrient absorption and decreasing the relative abundance of potentially pathogenic bacteria (Health and Nutritional Properties of Probiotics in Food Including Powder Milk with Live Lactic Acid Bacteria; Report of a Joint FAO/WHO Expert Consultation, Córdoba, Argentina, 1-4 Oct. 2001; FAO: Rome, 1-34; Amalaradjou et al., 2012, Adv. Food. Nutr. Res. 67, 185-239). Lactic acid bacteria (LAB), which include microbes in the genera Lactobacillus and Lactococcus, and Bifidobacterium, are currently the bacteria most commonly employed as probiotics (Oelschlaeger et al., 2010, Int. J. Med. Microbiol. 300, 57-62; Amalaradjou et al., 2012, Adv. Food. Nutr. Res. 67, 185-239). A number of probiotic bacteria are currently in use as nutritional supplements for humans and animals (Lodemann et al., 2006, Arch. Anim. Nutr. 60, 35-48; Lyra et al., 2010, BMC Gastroenterol. 10, 110; Percival et al., 1997, Clin. Nutr. Insights 6, 1-4; Whitley et al., 2009, J. Anim. Sci. 87, 723-728; Gerasimov et al., 2010, Am. J. Clin. Dermatol. 11, 351-361; Berman et al., 2006, Nutr. Res. 26, 454-459; Ahmed et al., 2007, J. Nutr. Health. Aging 11, 26-31). In addition, recombinant LAB are also significant therapeutic delivery vectors. They are presently being tested as candidates for the delivery of therapeutics inside the GI tract of humans for the treatment of inflammatory bowel syndrome and Crohn's disease (Braat et al., 2006, Clin. Gastroenterol. Hepatol. 4, 754-759; Dylaq et al., 2013, Curr. Pharma. Design 10; De Greef E et al., 2013, Acta Gastroenterol. Belg. 76, 15-9).